Organochlorine Burdens in Harbour Seals from the German Wadden Sea Collected During Two Phocine Distemper Epizootics and Ringed Seals from West Greenland Waters

Copyright: © 2012 Siebert U, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Organochlorine Burdens in Harbour Seals from the German Wadden Sea Collected During Two Phocine Distemper Epizootics and Ringed Seals from West Greenland Waters


Introduction
Organochlorines such as hexachlorocyclohexane (α-, β-and γ-HCH), hexachlorobenzene (HCB), dichlorodiphenyltrichlorethane and its metabolites (DDT, DDE and DDD), and polychlorinated biphenyls (PCB) accumulate in the marine ecosystem as shown in several studies (reports of AMAP, UNEP, TMAP). Negative effects were demonstrated for most of those chemicals, resulting in a European ban of DDT in 1972 and of PCBs in 1989, and a worldwide ban in 2001 (www.chem.unep.ch/pops).
Marine mammals are at the top of the marine food chain and accumulate chemical pollutants in their tissues. Ringed seals (Phoca hispida) are widely distributed in arctic and subarctic waters [1,2]. They feed on fish and crustaceans (Parathemisto sp.) and give birth to their offspring on ice in the spring [3]. Harbour seals (Phoca vitulina) are the most common pinniped species in the Wadden Sea. They feed mainly on fish in the open North Sea and give birth on the sand banks of the Wadden Sea during the summer months [4]. Two epizootics caused by the phocine distemper virus (PDV) occurred among the harbour seal population of the Wadden Sea and adjacent waters [5][6][7]. These outbreaks resulted in the deaths of more than 23,000 harbour seals in 1988 and 30,000 in 2002 [8][9][10].
Negative effects of organochlorines have been described for different seal species. Stenosis/occlusion of the uterus, osteoporosis, colonic ulcera, splenic atrophy, and lymphocytic depletion in thymus and lymph nodes were found in grey and ringed seals from the Baltic Sea and harbour seals from the Wadden Sea [11][12][13][14][15][16]. Immunological investigations revealed that the impairment of the immune response by organochlorines increased the susceptibility to diseases caused by viruses such as phocine distemper virus [17,18]. Therefore, an effect on the occurrence of epizootics in the harbour seal populations of the North and Baltic Seas could not be ruled out [8]. German samples were placed in aluminum foil and stored in a -18°C freezer until analysis. Because of limited sample size varying numbers of samples were available for individual PCB investigations.
For statistical analysis of the data the statistical open source platform R version 2.9.2 (R Development Core Team 2009) was used.
Testing for significant differences between the pollution levels in liver and blubber of the harbour seals, distinguished for PDV-positive and PDV-negative, a T-test was applied for simplicity as the values were more or less symmetrically distributed around the mean. As the number of animals with both liver and blubber data available was low, the results must be viewed with caution.
For comparison of the harbour seals data of from seal die-off in 1988 [21] and in 2002, only the mean values of the pollutant levels for the 1988 data were available. Here, a one-sample T-test was used.
The effect of age on the pollutant levels was tested using a regression analysis between the single pollution level as response and age as predictor, applying a GLM using Poisson error distribution with a correction of the standard errors using a quasi-GLM with a second order polynomial for males and females separately in combination with a backward selection. The 5% level of statistical significance was selected.
When comparing the pollution levels between ringed and harbour seals and within each population between liver and blubber, a general linear model (GLM) was applied using Poisson error distribution with a correction of the standard errors using a quasi-GLM [22,23], as the variance is different between both populations and, as mentioned earlier, the sample size is low. With a larger sample size with comparable variance structure, a GLM with log normal distribution would have been used.

Results and Discussion
Comparison between harbour seals found around the two seal epizootics Harbour seals were collected in and around the year 2002 when the second phocine distemper virus epizootic occurred [6,7]. 15 individuals were infected with phocine distemper virus as shown by PCR and immunocytochemistry [7]. Contrary to males, infected females showed higher concentrations of organochlorines in the liver than those who tested negative (Table 2, Figure 1). In liver tissue, the results were significant for DDE using a T-test (t = 2.93, p-value = 0.01, df = 10.99) and most of the PCBs (e.g. PCB 128: t = 2.49, p-value = 0.03, df = 10.081) congeners. In blubber tissue, however, results did not show significant differences. The correlation between PCBs concentrations and susceptibility to phocine distemper virus infections had been investigated through different research activities during the epizootics. [24] found higher organochlorine (OC) concentrations in the blubber of diseased harbour seals compared to those who survived the die-off. A feeding experiment showed that the immunosuppressive

Comparison between regions
All organochlorines analysed in the present study were found in seals from both areas (Table 4). In both species, hexachlorocyclohexane (α-, β-and γ-HCH), hexachlorobenzene (HCB), dichlorodiphenyltrichlorethane and its metabolites (DDT, DDE and DDD), and polychlorinated biphenyls (PCBs) were found. In blubber and liver of both species, PCBs 138 and 153 showed the highest concentrations, followed by PCBs 187, 180 and 170. The PCBs burden in blubber and liver of harbour seals was several times higher than in ringed seals (e.g., PCB 138) with the exception of PCB 52, which was highest in the liver of ringed seals. Levels of DDE and DDD were also higher in harbour seals (blubber -DDE: t = -7.94 p << 0.01, df = 37, DDD: t = -6.23 p << 0.01, df = 33) than in ringed seals, only DDT was higher in blubber of ringed seals (t = 2.61 p = 0.01, df = 35). α-, β-HCH and HCB in blubber were higher (e.g., α-HCH: t = 11.73 p << 0.01, df = 38) but in liver lower in ringed seals than harbour seals (e.g., HCB: t = -5.32 p << 0.01, df = 7). For γ-HCH concentrations, it was the opposite, with higher levels in liver of ringed seals (t = 2.81, p = 0.01, df = 13) and lower levels in blubber (t = -5.34, p << 0.01, df = 26). The variance within the ringed and harbour seals concerning the single pollutants was high (Table 4, e.g. PCB 52 or DDT). This could not be explained by the parameters of age or sex available in this study.
Levels found in Greenlandic ringed seals from the west coast were similar to those in ringed seals from the same area of Greenland, Alaska and Canada [25][26][27]. But levels were lower than those in ringed seals originating from the east coast of Greenland, Norway and Svalbard [26,28,29].

Temporal trends
From 1988 to 1992, the burdens of PCBs and DDT in harbour seals from German waters increased [21,30]. But when comparing findings between 1988-1992 [21,30] and 2002 (this study), concentration levels of all pollutants decreased, e.g. α-HCH and DDT by the factor 20 (Table 3 and 4, p << 0.05). Unfortunally no data on PCB levels of harbour seals from German waters are available for the period between 1992 and 2001 explaining when the decrease in body burden started. The decline in the burdens of HCB, HCH, OCS, DDT and DDD was distinct and confirmed earlier investigations [30]. A decreasing input can thus be assumed after the banning of technical HCH mixtures in many countries. The concentrations of PCBs and DDE decreased less noticeably because they are more persistent in the marine food web. Those findings were also confirmed in 18 harbour seals found dead in the Dutch Wadden Sea during the second PDV epizootic in 2002 [31]. HCH and HCB levels showed a significant decline but PCBs, DDT and DDE did not decrease significantly.
In all investigations PCBs 138 and 153 showed the highest concentrations. In general, 'legacy' persistent organic pollutants (POP) in arctic biota has showed decreasing trends during the past two to three decades [32]. This has also been the case for West Greenland ringed seals. For example, the concentrations of PCBs and DDT showed an annual decrease of 6.1 and 8.1%, respectively, during the period 1994 to 2006 [27].

Sex and age ratio
Male and female ringed and harbour seals in this study did not show a significant difference in PCBs using a GLM for testing (e.g., PCB 101, ringed seals: t = -1.13, p = 0.28, df = 18; harbour seals: t = 0.62 p = 0.54, df = 18), DDT (ringed seals: t = -0.57, p = 0.58, df = 18; harbour seals: t = -0.89, p = 0.39, df = 18) and α-, β-and γ-HCH concentrations ( Figure 2). In contrast, findings from other studies indicate that adult males carry higher burdens of any of these chemicals than females [21,26,33,34]. According to Reijnders [35], a plateau in concentrations is reached at about 4 years because intake and excretion are balanced. Mature female marine mammals lose organochlorines during gestation and lactation [36,38], resulting in a reduction of the organochlorine burden. In contrast, males continue to accumulate organochlorines throughout life [37]. In the present study, most of the seals were young; especially the ringed seals from West Greenland and, therefore, no difference between the sexes could be expected.
Age correlation was insignificant (not shown), most likely due to the short age gradient of the investigated seals and the low representation of seals of higher age (Table 1). Young harbour seals already showed higher organochlorine concentrations when compared to ringed seals, most likely due to a transfer of the burden from contaminated female harbour seals during lactation [38]. In harbour seals from the Dutch Wadden Sea, newborns showed an even higher PCB burden than subadults [33,35]. Birnbaum [39] and Debier et al. [37] showed for harbour and grey seals that higher chlorinated PCB congeners are transferred in higher concentrations through the milk than lower chlorinated ones.
In summary the present study showed that female harbour seals infected with phocine distemper virus carried higher concentrations of organochlorine confirming previous investigations from other areas [24]. All organochlorines analysed in this investigation were found in seals from German and Greenlandic waters. HCH and HCB levels showed a significant decline but PCBs, DDT and DDE did not Table 3: Mean values of pollutants for harbour seals 1992 [Vagts] (ng/g lipid weight), standard deviation is not available. The means were tested for significant differences by one-sample T-test with the data of 2002, the result is given in the column p; s. = significant different, n.s. = not significant different.  (Table 4). Pathological findings such as loss of bone structure, leiomyoma and occlusion of the uterus associated with high organochlorine burdens for ringed and grey seals from the Baltic [11,12,14] were not found in harbour seals from the North Sea [7,19]. However, concentrations of those substances are still at levels known to affect the immune and endocrine systems and to cause vitamin A deficiency in harbour seals and porpoises from the North Sea [17,18,[40][41][42]. Therefore, a contribution of organochlorines to the severity of mass mortalities and effects in seals with high burdens cannot be ruled out. Additionally, harbour seals from the Wadden Sea are also exposed to other pollutants such as perfluorooctane sulfonate (PFOS), polybrominated diphenyl ethers (PBDEs) and heavy metals [31,[43][44][45] as well as other effects of anthropogenic activities [4] so that continuous pressure on the health of the animals needs to be assumed and further investigations should be carried out.